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Biomedical subjects

A M Scanu

Publications and source records attributed to A M Scanu.

At least 145 records · Page 8Linked to original sources
PubMed1983

Proteolytic processing of human preproapolipoprotein A-I. A proposed defect in the conversion of pro A-I to A-I in Tangier's disease.

The primary translation product of human intestinal apolipoprotein A-I mRNA was isolated from wheat germ and ascites cell-free translation systems. Comparison of its NH2-terminal sequence with that of plasma high density lipoprotein-associated A-I showed that it is initially synthesized as a preproprotein. Like rat preproapolipoprotein A-I, it contains an 18-amino acid prepeptide and a 6-amino acid propeptide. The highly unusual COOH-terminal Gln-Gln dipeptide present in the rat pro-segment is also represented at the same position in the human sequence. The functional division of the 24-amino acid NH2-terminal extention into pro- and presegments was verified by finding that the stable intracellular form of A-I in a human hepatoma cell line was the proprotein. Edman degradation of radiolabeled intracellular and extracellular A-I indicated that this apolipoprotein was secreted without proteolytic cleavage of its hexapeptide prosegment. Therefore, it appears that apolipoprotein A-I undergoes an additional proteolytic processing step before it is fully integrated into plasma high density lipoprotein. Two-dimensional gel electrophoresis of purified proapolipoprotein A-I isolated from the hepatocyte cell culture media indicated that it corresponds to isoforms 2 and 3, the basic A-I isoproteins which are the precursors of plasma A-I and the predominant plasma A-I isoforms found in patients with Tangier's disease (Zannis, V. I., Lees, A. M., Lees, R. S., and Breslow, J. L. (1982) J. Biol. Chem., 257, 4978-4986). Therefore this pathologic state probably arises from a defect in the conversion of proapolipoprotein A-I to apolipoprotein A-I.

Amino Acid Sequence↗
PubMed1983

Isolation and characterization of human apolipoprotein A-IV from lipoprotein-depleted serum.

Human apolipoprotein A-IV is an acidic polypeptide of molecular weight 46,000 that is secreted into lymph on the surface of nascent chylomicrons, but which exists in circulation unassociated with lipoproteins. Previous studies of this protein have utilized material isolated from a d < 1.006 g/ml fraction of human serum and from human lymph. Although it has been suggested that apoA-IV circulating in plasma is the product of dissociation from the surface of nascent chylomicrons, it has been characterized by immunological techniques only. We have isolated human apoA-IV on a preparative scale from lipoprotein-depleted serum using a technique of adsorption to a phospholipid-triglyceride emulsion, followed by delipidation and preparative gel electrophoresis. The molecular weight, pI, and amino acid composition of material thus prepared agree with literature values for apoA-IV derived from chylomicrons. We have determined that apoA-IV is a glycoprotein containing 6% carbohydrate by weight (mannose 1.8%, galactose 1.55%, N-acetyl glucosamine 1.55%, sialic acid 1.1%). Electroimmunoassay of human serum using a monospecific rabbit antibody to serum-derived apoA-IV found 13.1 +/- 1.8 mg/dl, a value in agreement with determinations using antibodies to chylomicron-derived apoA-IV. We conclude that apoA-IV may be easily purified from normal human serum, and that the material thus isolated has identical chemical, physical, and immunological properties to apoA-IV obtained from human lymph. It is therefore likely that the dissociation of apoA-IV from the surface of nascent chylomicrons following their entry into circulation is not attended by changes in the structure or composition of this apoprotein.-Weinberg, R. B., and A. M. Scanu. Isolation and characterization of human apolipoprotein A-IV from lipoprotein-depleted serum.

Amino Acids↗
PubMed1983

Rapid isolation of apolipoprotein E from human plasma very low density lipoproteins by molecular sieve high performance liquid chromatography.

A rapid (less than 1 hour) and sensitive technique was developed for the isolation of apolipoprotein E from the other main components of human plasma very low density lipoproteins using molecular sieve high performance liquid chromatography with an approximate 80% recovery. The properties of the pure apoprotein were those reported in the literature for products isolated by conventional chromatographic and/or electrophoretic procedures.

Apolipoproteins↗
PubMed1982

Organization of unesterified cholesterol in high density lipoproteins probed by filipin.

The initial rate of filipin association with unesterified cholesterol in high density lipoproteins (HDL) was measured by stopped-flow spectrophotometry to assess the roles played by apolipoproteins and phospholipids in modulating the surface exposure of cholesterol. The initial rate of filipin-unesterified cholesterol association was enhanced upon hydrolysis of the glycerophospholipids of human HDL3 by phospholipase A2. Rate enhancements were also observed following trypsin-catalyzed hydrolysis of apolipoprotein A-I in canine HDL and of apolipoproteins A-I and A-II in human HDL3. However, the initial rate of filipin-unesterified cholesterol association was not altered upon incubation of HDL3 with polymorphonuclear cells, which causes hydrolysis of apolipoprotein A-II but leaves apolipoprotein A-I intact. These results are consistent with the general structural model of HDL in which unesterified cholesterol, apolipoproteins and glycerophospholipids are presumed to be localized at the surface of the HDL particle. From these studies and from results indicating that the initial rate of filipin-unesterified association was enhanced in canine HDL hybrids in which 50% of the apolipoprotein A-I had been replaced by apolipoprotein A-II, we also conclude that apolipoprotein A-I in HDL is in closer proximity to unesterified cholesterol than apolipoprotein A-II. Thus, it appears that rapid kinetic measurements of filipin-cholesterol association may be useful in assessing the organization of unesterified cholesterol in serum lipoproteins.

Animals↗
PubMed1982

Properties and metabolic fate of two very low density lipoprotein subfractions from rhesus monkey serum.

Physical, chemical and physiological approaches were used to examine the properties of two very low density lipoproteins, VLDL-I (slow-beta), and VLDL-II (pre-beta), which were isolated by agarose column chromatography from the serum of rhesus monkeys fed either Purina Chow or one of four hyperlipidemic diets containing 0.5-20% cholesterol suspended in either coconut oil, peanut oil, mixed coconut oil and butter fat or lard. In the coconut oil-fed hyperlipidemic animals, the majority of the apolar lipids of VLDL-I was represented by cholesteryl esters. The small percentage of triacylglycerol (15%) had a fatty acid composition which resembled that of the fatty acid in each of the diets. In turn, VLDL-II had a triacylglycerol-rich core and differed from VLDL-I in apolipoprotein distribution (VLDL-I: low molecular weight apolipoprotein B, 36%; apolipoprotein E, 64%; and VLDL-II: high molecular weight apolipoprotein B, 38%; apolipoprotein E, 3%; and apolipoprotein C, 65%). Both VLDLs were hydrolyzed in vitro by milk lipoprotein lipase by first-order kinetics although VLDL-I exhibited a slightly slower reaction rate. When an oral dose of [3H]retinol was given to one of the animals, both VLDLs became labeled but the specific activity of VLDL-I was six times higher than that of VLDL-II and the other lipoproteins. We conclude that VLDL-I represents a cholesteryl ester-rich lipoprotein probably of intestinal origin, whereas VLDL-II may be a particle of hepatic derivation modified by its interaction with the other plasma lipoproteins.

Animals↗
PubMed1982

The selective lipid-lowering effect of vegetarianism on low density lipoproteins in a cross-over experiment.

In a cross-over experiment the effect of short-term vegetarianism on serum lipids, lipoproteins and apoproteins was studied. The experimental diet was free of animal products, with the exception of skim milk, and consequently low in saturated fat and cholesterol. Fifteen free-living individuals were randomly assigned to 3-week periods on either the experimental diet or a control diet which closely approximated the usual intake in the U.S.A. Significant reductions in total cholesterol (12.5%), low density lipoprotein cholesterol (14.7%), and apo B (13.2%) were observed, accompanied by a non-significant decrease in high density lipoprotein cholesterol (10%), apo A-I (3%) and a non-significant increase in apo-II (4%). These data suggest that a fat-modified diet low in total fat, saturated fat and cholesterol, and moderate (not high) in polyunsaturated fat may not lower HDL-C or its apoproteins as much as a diet high in polyunsaturated fat, while having similar effects on LDL-C, and would therefore be preferable as the basis for primary prevention of atherosclerosis.

Adult↗
PubMed1982

In vitro reciprocal exchange of apoproteins and nonpolar lipids between human high density lipoproteins and an artificial triglyceride-phospholipid emulsion (Intralipid).

To determine the nature of lipid and apoprotein exchange between human high density lipoproteins (HDL) and Intralipid particles of Sf greater than 400 (ILIP) we have studied their in vitro interaction during incubation in aqueous buffer and in lipoprotein-deficient serum (LPDS). We found that ILIP acquires apo A-I, apo A-IV and apo E from LPDS, and that this uptake is inhibited by the presence of HDL, which readily donate C-apoproteins to the ILIP surface. In the absence of LPDS exchange of only polar lipids occurred between ILIP and HDL, with HDL gaining phospholipid from, and donating free cholesterol to this fat emulsion. In the presence of LPDS the exchange of nonpolar lipids occurred between the two particles: in the case of HDL, cholesteryl ester content decreased, accompanied by an increase in triglyceride, causing a decrease in the hydrated density of the lipoprotein and an increase in its molecular weight; in the case of ILIP, reciprocal changes in lipid content were seen as a loss of triglyceride and the appearance of cholesteryl esters. When compared to literature data, our findings indicate that Intralipid Sf greater than 400 particles exhibit an in vitro behavior which is remarkably similar to that of nascent chylomicrons with respect to the exchange of A- and C-apoproteins and surface polar lipids with HDL. We postulate that since ILIP and HDL can participate in a LPDS-dependent exchange of non-polar core lipids, that this process may occur when this fat emulsion is administered in vivo.

Apoproteins↗
PubMed1982

In vitro mass: activity distribution of lecithin--cholesterol acyltransferase among human plasma lipoproteins.

In order to compare the mass-activity distribution of lecithin-cholesterol acyltransferase (LCAT) among plasma lipoproteins separated by various ultracentrifugal or chromatographic procedures, we have quantified the enzyme by an electroimmunoassay technique using a specific antibody raised in the rabbit. This antibody, when added to whole serum, inhibited all of the enzyme activity present in it. The percent mass distribution of the enzyme among the lipoproteins isolated by rate-zonal ultracentrifugation (d 1.00-1.36 g/ml, SW 40 rotor, 37 000 rpm, 16 h) was as follows: very low density lipoproteins (VLDL), 0; low density lipoproteins (LDL), 6.2; HDL2, 6.5; HDL3, 12 and d greater than 1.21 g/ml fraction, 75. Measurement of LCAT activity of each lipoprotein fraction against mixed single bilayer lecithin-cholesterol vesicles (molar ratio, 4:1) containing apo A-I, indicated that VLDL, LDL and HDL2 were inactive or minimally active under the experimental conditions used, whereas HDL3 and the d greater than 1.21 g/ml fraction contained 17.5 and 79.9% of the total enzyme activity. Prolonged ultracentrifugation of the LCAT-containing lipoproteins resulted in the recovery of activity in the lipoprotein-free infranatant. In studies with lipoproteins linked to Sepharose 4B, LCAT was found to bind LDL, HDL2, and HDL3. It is concluded that LCAT is present in all the major lipoproteins except for VLDL. The activity appears to be dependent, at least in part, on the type of lipoproteins to which the enzyme is associated with.

Centrifugation, Isopycnic↗
PubMed1982

Serum low density lipoproteins with mitogenic effect on cultured aortic smooth muscle cells.

Low density lipoprotein (LDL) subspecies of different size and lipid mass were isolated by density gradient ultracentrifugation from the serum of male rhesus monkeys (Macaca mulatta) fed both a low fat, low cholesterol commercial primate ration, and cholesterol-supplemented high-fat diets, as well as from the serum of human donors. The mitogenic effect of these lipoproteins was examined using primary cultures of rhesus aortic smooth muscle cells. It was observed that the smaller LDL (molecular weight 2.7 X 10(6) from normolipidemic monkeys and a small LDL (molecular weight 2.6 X 10(6) occurring in some normal human subjects exhibited no mitogenic action. In turn, the larger LDL subspecies (molecular weight greater than 3.0 X 10(6), and buoyant density less than 1.030 g/ml), whether from normolipidemic or hyperlipidemic monkeys, or from some normal human subjects, had a marked proliferative action. The results indicate that both hyperlipidemic and normal sera (both human and rhesus) contain mitogenic LDL species although in different amounts. LDL-III, the rhesus equivalent of human Lp(a) was not mitogenic despite its similarity on size and lipid composition to the stimulating particles. However, on the removal of most of its large sialic acid moiety, a clear mitogenic action was observed. The mechanisms responsible for the proliferative effect are unclear and may involve LDL mass, lipid composition, and surface charge although other speculations cannot at present be ruled out. Furthermore, since the small LDL subspecies of either rhesus or human origin were nonmitogenic and similar in mass to the LDL found in calf serum, the mitogenic response of the smooth muscle cells to large LDLs may depend on their early conditioning with the LDL of calf serum.

Animals↗
PubMed1982

Normal serum and lipoprotein-deficient serum give different expressions of excitability, corresponding to different stages of differentiation, in chicken cardiac cells in culture.

Monolayers of cardiac cells from 11-day-old chicken hearts have different properties when maintained in fetal calf serum or in a lipoprotein-deficient serum (LPDS). Cells in fetal calf serum have a resting potential near -60 mV; the rate of rise of the action potential is low (less than 10 V/sec); the action potential and the contraction are essentially unaffected by tetrodotoxin (TTX); and the beating properties are unaffected by muscarinic agents. Cells in LPDS have a resting potential near -75 mV, and a fast rise of the action potential (approximately equal to 100 V/sec) that is drastically decreased by TTX with a parallel abolition of contraction, and the beat is blocked by very low concentrations of muscarinic agonists. Cells that are physiologically fully responsive to TTX and to muscarinic agents have receptors that remain stable 24 hr after protein synthesis is blocked, whereas cells that are physiologically unresponsive to TTX and muscarinic agents have receptors that are rapidly degraded with half-lives between 9 hr (TTX receptor) and 14 hr (muscarinic receptor). Differences in the physiological and biochemical properties are accompanied by changes in the cholesterol contents of the cell membranes. The properties of cardiac cells cultured in normal serum are similar to those found for cells of chicken hearts in the very early embryonic stage, whereas those of cardiac cells cultured in LPDS correspond to the late embryonic stage.

Animals↗
PubMed1982

Neutrophils are required for the DNA synthetic response of human lymphocytes to mevalonic acid: evidence suggesting that a nonsterol product of mevalonate is involved.

Human peripheral blood lymphocytes, in the presence of human neutrophils, initiate DNA synthesis and cell cycling when exposed to mevalonic acid. The ability of lymphocytes to respond in this manner is a radiosensitive property of cells, whereas the help provided by neutrophils is maintained despite their exposure to x-irradiation. Other organic acid anions, including precursors of mevalonic acid biosynthesis and a variety of products of mevalonate metabolism, fail to initiate DNA synthesis when added to human lymphocytes. Because only the metabolically active R(--) enantiomer of mevalonic acid initiates lymphocyte DNA synthesis, we presume that physiological pathways of mevalonate metabolism are involved. The response to mevalonic acid of ML-236B (compactin)-inhibited lymphocytes is increased, and the threshold concentration of meyalonate at which lymphocyte DNA synthesis first appears is decreased, when the cells are cultured in lipoprotein-containing (as opposed to lipoprotein-depleted) medium. The response to mevalonic acid of lymphocytes cultured in lipoprotein-depleted medium can be enhanced by addition to the cultures of low density lipoprotein but not by addition of high density lipoprotein. Based upon the flux diversion hypothesis of mevalonate metabolism, these observations suggest that a nonsterol product of mevalonate metabolism may be responsible for the initiation of lymphocyte DNA synthesis by mevalonic acid.

Cell Cycle↗
PubMed1981

Serum lipoproteins modulate oxygenated sterol insertion into human red cell membranes.

The insertion of oxygenated sterol compounds into human red blood cell membranes as well as the consequent transformation of the red cells to an echinocyte shape and the expansion of the membranes are impeded by the presence of serum lipoproteins in the incubation medium. All density classes of human serum lipoproteins bind oxygenated sterol compounds, and lipoproteins can act as acceptors of oxygenated sterols previously inserted into red cells. Since oxygenated sterols have been reported to be atherogenic, the modulating and possibly protective effects of serum lipoproteins on oxygenated sterol-induced derangement of cell membrane structure and function may provide a useful model for further study.

Cholesterol↗